Nonlinear imaging using passive pulse splitters and related technologies

ABSTRACT

An apparatus includes a pulsed laser source that produces a pulsed laser beam at an input repetition rate and an input pulse power, a passive pulse splitter that receives the pulsed laser beam and outputs a signal including a plurality of sub-pulses for each input pulse of the pulsed laser beam, a sample, and a detector. The output signal has a repetition rate that is greater than the input repetition rate and the powers of each of the sub-pulses are less than the input pulse power. The sample is placed in the path of a sample beam that is formed from the beam that exits the pulse splitter. The detector receives a signal of interest emitted from the sample.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/987,698, filed on Nov. 13, 2007, and of U.S. Provisional ApplicationNo. 60/971,792, filed on Sep. 12, 2007. Both of these applications arehereby incorporated by reference in their entirety.

TECHNICAL FIELD

The disclosure relates to passive pulse splitters and pulse splittersystems that include passive pulse splitters.

BACKGROUND

Pulsed lasers are used in nonlinear bio-imaging techniques such asmulti-photon fluorescence excitation microscopy (MPFM) or two-photonfluorescence excitation microscopy (2PFM), which is a type of MPFM. In2PFM, two photons of the same or different energy are absorbed by amolecule and the fluorescence from the molecule upon relaxation iscollected with a highly sensitive detector such as a photomultipliertube. Because the probability of near simultaneous absorption of twophotons is low, a high flux of excitation photons is usually needed, andtherefore an ultrafast (for example, femtosecond) pulsed laser istypically used. An example of a suitable pulsed light source is aTi:sapphire laser, which can operate from 650-1100 nm with pulse widthsof about 100-150 fs, repetition frequencies of about 70-80 MHz, and atseveral Watts of power (for example, 3 W).

SUMMARY

In some general aspects, a pulse splitting apparatus includes a pulsedlaser source that produces laser pulses at an input repetition rate andan input pulse power, and a pulse splitter that receives the laserpulses from the pulsed laser source and outputs a plurality ofsub-pulses for each laser pulse from the pulsed laser source. Thesub-pulses have a repetition rate that is greater than the inputrepetition rate and the powers of the sub-pulses are less than the inputpulse power. The pulse splitter includes at least two differentmaterials through which the sub-pulses travel such that each of thesub-pulses travel along different optical path lengths through thedifferent materials to cause a temporal separation between each of thesub-pulses. The optical path length depends on both the distancetraveled through the material and the index of refraction of thematerial.

Implementations can include one or more of the following features. Forexample, the pulsed laser source can be a Ti-Sapphire laser. The pulsedlaser source can produce time-averaged output powers of several Watts.The pulsed laser source can operate with an input repetition rate ofabout 70-80 MHz, a pulse width from about 10 fs to about 2 ps, and at a650-1100 nm wavelength.

The pulse splitter can include one or more pairs of at leastpartly-reflecting surfaces that face each other, a first region betweenthe one or more surface pairs and having a first refractive index and afirst thickness, a second region between the one or more surface pairsand adjacent the first region, and having a second refractive index thatis different from the first refractive index and a second thickness thatis different from the first thickness, and a partially reflectiveinterface between the first and second regions. The pulse splitter canoutput N sub-pulses for every one pulse input to the pulse splitter.

The pulse splitting apparatus can include a recombiner that receives theplurality of sub-pulses from the pulse splitter and recombines thesub-pulses to form a sub-pulse light source that is directed to asample. The pulse splitting apparatus can include a delay external tothe pulse splitter and through which at least some of the output of thepulse splitter is passed. The pulse splitting apparatus can include amotion stage to which the pulse splitter is attached such that the pulsesplitter can be moved relative to the path that the laser pulses travelalong.

The pulse splitter can be a passive pulse splitter.

The plurality of sub-pulses can be of equal or approximately equalintensity.

The pulse splitter can output the plurality of sub-pulses usingsubstantially all of the available power from the pulsed laser source.

The pulse splitting apparatus can include a sample placed in the path ofa sample beam that is formed from an output of the pulse splitter, and adetector that receives a multi-photon fluorescence signal emitted fromthe sample. The pulse splitting apparatus of claim 13, wherein thetemporal separation between sub-pulses should be large enough to enablea molecule of the sample to relax to a lowest vibrational state of itsexcited state that is reached after multiple photons have been absorbedby the molecule.

Each of the sub-pulses can have a power that is less than the inputpulse power. The repetition rate of the sub-pulses can vary.

In another general aspect, a method of pulse splitting includesproducing a pulsed laser beam having a repetition rate and a pulsepower, directing the pulsed laser beam into a pulse splitter thatcreates a finite number of sub-pulses for each input pulse of the pulsedlaser beam by directing sub-pulses through at least two differentmaterials and along different optical path lengths through the differentmaterials to cause temporal separation between each of the sub-pulses.The pulse splitter produces an output signal that has a repetition ratethat is greater than the repetition rate of the pulsed laser beam, andthe powers of the sub-pulses are less than the pulse power of the pulsedlaser beam.

Implementations can include one or more of the following features. Forexample, the pulsed laser source can be a Ti-Sapphire laser. The pulsedlaser source can produce time-averaged output powers of several Watts.The pulsed laser source can operate with an input repetition rate ofabout 70-80 MHz, a pulse width from about 10 fs to about 2 ps, and at a650-1100 nm wavelength.

The pulsed laser beam can be directed into the pulse splitter bydirecting the pulsed laser beam into a first region that is definedbetween first and second reflecting surfaces and has a first refractiveindex and a first thickness such that the laser beam travels through thefirst region and impinges upon a partially reflective interface betweenthe first region and a second region adjacent the first region, and issplit at the partially reflective interface, where the light that isreflected from the partially reflective interface strikes the firstreflecting surface and any light that transmits through the partiallyreflective interface strikes the second reflecting surface. The pulsesplitter can output N sub-pulses for every one pulse input to the pulsesplitter and the partially reflective interface is configured to provideN passes or bounces of the light through the pulse splitter.

The method can include recombining the equal intensity sub-pulses outputfrom the pulse splitter to form a sub-pulse light source that isdirected to a sample. The method can include directing at least some ofthe sub-pulses output from the pulse splitter through a delay externalto the pulse splitter. The method can include adjusting a position ofthe pulse splitter relative to the laser beam path.

The pulse splitter can produce the output signal without using externalenergy to act on the pulsed laser beam.

In another general aspect, a passive pulse splitter includes one or morepairs of at least partly-reflecting surfaces that face each other; afirst region between the one or more surface pairs, the first regionhaving a first refractive index and a first thickness; a second regionbetween the one or more surface pairs, the second region being adjacentthe first region, and having a second refractive index that is differentfrom the first refractive index, a second thickness that is differentfrom the first thickness, or both a second refractive index that isdifferent from the first refractive index and a second thickness that isdifferent from the first thickness; and a partially reflective interfacebetween the first and second regions.

Implementations can include one or more of the following features. Forexample, the second region can be defined as an open air-filled regionbetween the pair of at least partly-reflecting surfaces. The firstregion can be a fused silica substrate having a first surface thatincludes a coating that forms one of the at least partly-reflectingsurfaces and having a second opposite surface that includes a coatingthat forms the partially reflective interface.

The partially reflective interface can include two or more segments ofdifferent reflectance. The partially reflective interface can include atleast one segment that is 100% reflective.

In another general aspect, an apparatus includes a pulsed laser sourcethat produces a pulsed laser beam at an input repetition rate and aninput pulse power; a passive pulse splitter that receives the pulsedlaser beam and outputs a signal including a plurality of sub-pulses foreach input pulse of the pulsed laser beam, a sample placed in the pathof a sample beam that is formed from the beam that exits the pulsesplitter; and a detector that receives a signal of interest emitted fromthe sample. The output signal of the pulse splitter has a repetitionrate that is greater than the input repetition rate and the powers ofeach of the sub-pulses are less than the input pulse power.

Implementations can include one or more of the following features. Thedetector can receive signals of interest from a plurality of locationsat the sample to form an image. The signal of interest can bemulti-photon fluorescence emitted from a nonlinear biological sample.

The pulsed laser source can be a Ti-Sapphire laser. The pulsed lasersource can produce time-averaged output powers of several Watts. Thepulsed laser source can operate with an input repetition rate of about70-80 MHz, a pulse width from about 10 fs to about 2 ps, and at a650-110 nm wavelength.

The signal of interest can be a second harmonic generation signalemitted from a nonlinear sample. The signal of interest can be a signalemitted from a nonlinear sample using coherent anti-Stokes Ramanscattering spectroscopy with the output signal from the passive pulsesplitter.

The pulse splitter can include one or more beam splitters and one ormore beam couplers. The pulse splitter can include a plurality ofwaveguides.

The pulse splitter can include at least two different materials throughwhich the sub-pulses travel such that each of the sub-pulses travelalong different optical path lengths through different materials tocause temporal separation between each of the sub-pulses, wherein theoptical path length depends on both the distance traveled through thematerial and the index of refraction of the material.

The pulse splitter can include one or more pairs of at leastpartly-reflecting surfaces that face each other; a first region betweenthe one or more surface pairs and having a first refractive index and afirst thickness; a second region between the one or more surface pairsand adjacent the first region, and having a second refractive index thatis different from the first refractive index and a second thickness thatis different from the first thickness; and a partially reflectiveinterface between the first and second regions.

The pulse splitter can output N sub-pulses for every one pulse input tothe pulse splitter.

The apparatus can include a recombiner that receives the plurality ofsub-pulses from the pulse splitter and recombines the sub-pulses to formthe sample beam. The apparatus can include a delay external to the pulsesplitter and through which at least some of the output of the pulsesplitter is passed. The apparatus can include a motion stage to whichthe pulse splitter is attached such that the pulse splitter can be movedrelative to the path that the laser pulses travel along.

The plurality of sub-pulses can be of equal or approximately equalintensity.

The pulse splitter can output the plurality of sub-pulses usingsubstantially all of the available power from the pulsed laser source.

In another general aspect, a signal detection method includes producinga pulsed laser beam having a repetition rate and a pulse power;directing the pulsed laser beam into a passive pulse splitter thatcreates a finite number of sub-pulses for each input pulse of the pulsedlaser beam and produces an output signal that has a repetition rate thatis greater than the repetition rate of the pulsed laser beam, directinga sample signal produced from the output signal of the pulse splitter toa sample; and directing a signal of interest emitted from the sample dueto the interaction of the sample with the pulse splitter output to adetector. The powers of the sub-pulses are less than the pulse power ofthe pulsed laser beam;

Implementations can include one or more of the following features. Forexample, the method can include directing signals of interest from aplurality of locations at the sample to the detector to form an image.The method can include detecting the signal of interest emitted from thesample. The signal of interest emitted from the sample can be detectedby detecting a fluorescence signal from the sample.

The method can include recombining sub-pulses from the pulse splitteroutput signal to form the sample signal. The method can includesplitting the pulse splitter output signal into at least two signals anddelaying at least one of the signals relative to the other signal.

The pulse splitter creates a finite number of sub-pulses from each inputpulse, and does so with minimal or reduced power loss, so that the fullpower of the laser can be used to achieve larger (for example, greaterthan 100×) repetition rate gains at sub-pulse powers comparable to thesingle pulse powers currently used at the samples to be imaged. In someimplementations, the sub-pulses can have equal or approximately equalpowers. Such a design can be advantageous to prevent more powerfulpulses from dominating the signal and causing photo-bleaching or damage.In other implementations, the sub-pulses can have different powers.Furthermore, the pulse splitter does not require undue manual alignment,even for the design in which the sub-pulses co-propagate to within about10 arcsec in order for them to reach a common focal point within thesample, to obtain desired spatial resolution.

The pulse splitter is compact, requires few adjustments, and can beadded to or retrofit to existing pulsed laser or microscope used in 2PFMwithout undue effort. The pulse splitter can be flexibly reconfigured toachieve desirable repetition rate, pulse spacing, and pulse intensitybased on the photophysics of the system under investigation. The pulsesplitter does not introduce unacceptable dispersion, permits repetitionrate gains of at least 100×, and is adaptable to the large installedbase of pre-existing lasers and 2PFM microscopes.

By coaxing higher signal rates from a single focus, the pulse splitteris well suited for data acquisition rates useful in a host of nonlinearoptical applications including, but not limited to, neuroscience, andlinear optical applications like Raleigh scattering or communications.If reduced photoinduced bleaching and damage is important, then pulsesplitting using the pulse splitter can greatly increase the totalintegrated signal obtainable from various sample preparations, rangingfrom green fluorescent protein (GFP) in fixed tissues and living C.Elegans larvae, to Ca²⁺ indicators in acute brain slices.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of an imaging apparatus that includes a pulsedlaser source that produces a pulsed laser beam;

FIGS. 2A and 2B are top cross-sectional diagrams of a pulse splitterthat can be used in the imaging apparatus of FIG. 1;

FIG. 3A is a top cross-sectional view of a pulse splitter that producesoutput pulses having an intra-pair pulse spacing of 37 ps and isdesigned based on the pulse splitter of FIGS. 2A and 2B;

FIG. 3B is a side perspective view of the pulse splitter of FIG. 3A;

FIG. 4A is a top cross-sectional view of a pulse splitter that producesoutput pulses having an intra-pair pulse spacing of 74 ps and isdesigned based on the pulse splitter of FIGS. 2A and 2B;

FIG. 4B is a side perspective view of the pulse splitter of FIG. 4A;

FIG. 5A is a diagram of a pulse splitter system including a pulsesplitter that is designed based on the pulse splitter of FIGS. 2A and2B;

FIG. 5B is a diagram of a pulse shape output by the pulse splittersystem of FIG. 5A;

FIG. 6 is a top cross-sectional diagram of another implementation of apulse splitter that is designed based on the pulse splitter described inFIGS. 2A and 2B;

FIG. 7 is a top cross-sectional diagram of another implementation of apulse splitter that is designed based on the pulse splitter described inFIGS. 2A and 2B;

FIG. 8 is a diagram of a pulse splitter system including pulse splittersthat are designed based on the pulse splitter of FIGS. 2A and 2B;

FIGS. 9A-9E are diagrams of pulse splitter systems;

FIGS. 10A-10E are diagrams showing timing of output pulses from thepulse splitters of, respectively, FIGS. 9A-9E;

FIGS. 11A, 11D, and 11G are images of brain slices measured with a pulsesplitter system and with different operational parameters;

FIGS. 11B, 11E, and 11H are images of brain slices measured without apulse splitter system and with different operational parameters;

FIGS. 11C, 11F, and 11I are graphs showing comparative line profilestaken along the arrows shown at left images with a pulse splitter (inFIGS. 11A, 11D, and 11G) and center images without a pulse splitter(FIGS. 11B, 11E, and 11H);

FIG. 12A is a graph of a ratio of the average power to obtain aparticular signal rate with and without a pulse splitter system;

FIG. 12B is a graph of photobleaching versus time with and without apulse splitter system of GFP-labeled mouse brain slices;

FIG. 12C is a graph of summarized ratios of photobleaching decay timeswith and without a pulse splitter system for five different pulsesplitter systems of FIGS. 9A-9E;

FIGS. 13A and 13B are images of muscle cells of GFP-labeled C. Eleganslarva taken without a pulse splitter system at a time immediatelyfollowing laser exposure (FIG. 13A) and at a time 53 s after laserexposure (FIG. 13B);

FIGS. 13C and 13D are images of muscle cells of GFP-labeled C. Eleganslarva taken with a pulse splitter system of FIG. 9D at a timeimmediately following laser exposure (FIG. 13C) and at a time 120 safter laser exposure (FIG. 13D);

FIG. 13E is a graph of a normalized fluorescence signal versus timeobtained with repeated line scans over single muscle cells with andwithout the pulse splitter system;

FIG. 14A is a graph of an un-normalized signal versus time obtained withthe pulse splitter system of FIG. 9D;

FIG. 14B is a graph of an un-normalized signal versus time obtainedwithout a pulse splitter system;

FIGS. 15A, 15B, and 15C are graphs of normalized fluorescence signalchange versus number of traces and exposure time as measured from twodendritic branches of a single neuron, one with a pulse splitting systemof FIG. 9D (the top green circles) and one without a pulse splittingsystem (the bottom grey circles);

FIGS. 15D and 15E are basal fluorescence images taken before (FIG. 15D)and after imaging (FIG. 15E) of the dendritic branches from which thedata in FIGS. 15B and 15C, respectively, were obtained;

FIGS. 16A-16C are graphs of signal versus time for each series of tracesof FIGS. 15A-15C, respectively;

FIGS. 17 and 18 are top cross-sectional diagrams of pulse splitters thatcan be used in the imaging apparatus of FIG. 1; and

FIG. 19 is a diagram of a pulse splitter that uses optical waveguidesand that can be used in the imaging apparatus of FIG. 1.

DETAILED DESCRIPTION

A characteristic common to multi- or two-photon fluorescence microscopy(2PFM) measurements is the small excitation cross-sections involved,resulting in weak signals, long data acquisition times, and highrequisite pulse powers. Furthermore, such signals cannot be increasedarbitrarily by increasing pulse power, because in many casesphotoinduced damage of the sample increases even faster with power.Thus, only a fraction of the laser power available can be delivered tothe sample before photo-induced damage becomes excessive. For example,although most common ultrafast systems (that is, Ti:Sapphire lasersystems) offer time-averaged output powers of several Watts, 2PFMimaging in a biological context is usually limited by photodamage toaverage powers of less than 10 mW at the sample, often yielding lessthan one detected photon per pulse, and wasting over 99% of theavailable laser power.

Referring to FIG. 1, an imaging apparatus 100 includes at least onepulsed laser source 105 that produces a pulsed laser beam 107 at aninput repetition rate and an input pulse power, an optical arrangement110 that includes one or more passive pulse splitters 112, a nonlinearbiological sample 115 to be imaged that is placed in the path of a beam120 that exits the passive pulse splitter 110, and a detector 125 thatreceives a signal of interest emitted from the sample 115. The passivepulse splitter 112 receives the pulsed laser beam 107 from the lasersource 105 or from another component within the optical arrangement 110,and outputs a signal including a plurality of equal or non-equal powersub-pulses for each input pulse of the pulsed laser beam 107, where theoutput signal has a repetition rate that is greater than the inputrepetition rate and the powers of the sub-pulses are less than the inputpulse power. The apparatus 100 may also include other beam shaping anddirecting optics 130-132 for guiding the beams 107, 120 to the sample115 or for guiding the signal to the detector 125 and the sample 115 canbe mounted on a sample accommodating structure such as a suitable mount135. Thus, the sample 115 can therefore receive the beam output directlyfrom the pulse splitter 112 or can receive a sample beam that is formedfrom the beam that is output directly from the pulse splitter 112.

As discussed below, the passive pulse splitter 112 is designed toconvert each laser pulse from the laser source 105 into a plurality ofsub-pulses having equal, approximately equal, or unequal power, and thepulse splitter 112 can be designed to produce a particular sub-pulserepetition rate and a total number of sub-pulses. Thus, each of thesub-pulses can have a power that is similar to the powers of the othersub-pulses or the power of each of the sub-pulses can vary within asuitable range. Moreover, the repetition rate can vary along the trainof the pulses within a suitable range. The pulse splitter 112 is“passive” in that it requires no active components that would use energyto act on the laser pulses. Examples of designs for the pulse splitter112 are described below and shown in the remaining drawings.

To demonstrate its use, as discussed below, the pulse splitter 112 isapplied to 2PFM imaging of fixed brain slices labeled with greenfluorescent protein (GFP) and in different power regimes, the pulsesplitter can either be used to increase the signal rate more than 100fold, or to reduce the rate of photobleaching by over four-fold. Inliving specimens, the gains can be even greater: a nine-fold reductionin photobleaching during in vivo imaging of C. Elegans larvae; and a sixto twenty-fold decrease in the rate of photodamage during functionalcalcium imaging of hippocampal brain slices.

One way to increase the signal rate and hence the speed of dataacquisition N-fold is to accelerate the pulse repetition rate N-foldwhile maintaining the original pulse power. This can yield reducedphotodamage compared to the approach of increasing the pulse power whilemaintaining the original repetition rate. In another limit, detailedbelow, the rate of photodamage at constant signal can be decreased byincreasing pulse repetition rate, provided that the power per pulse isdecreased appropriately.

Referring to FIG. 2A, a pulse splitter 200 includes a pair of reflectingsurfaces 205, 210 that are parallel with and face each other to definean internal opening. The reflecting surfaces 205, 210 can be planar todefine a rectangular internal opening. The reflecting surfaces 205, 210can be mirrored surfaces.

The pulse splitter 200 includes a first region 215 between the pair ofreflecting surfaces 205, 210 and a second region 220 adjacent to thefirst region 215 and between the first region 215 and the reflectingsurface 210. The first region 215 has a first refractive index n₁ and afirst thickness d₁. The second region 220 has a second refractive indexn₂ that is different from the first refractive index n₁ and a secondthickness d₂ that is different from the first thickness d₁. The pulsesplitter 200 includes a partially reflective interface 225 between thefirst and second regions 215, 220.

The regions 215, 220 are parallel with each other and are sandwichedbetween the two reflecting surfaces 205, 210. The partially reflectiveinterface 225 can be a partially reflective coating applied to one ormore of the materials that make up the regions 215, 220 at the interfacebetween the two regions 215, 220.

In one implementation shown in FIG. 2A, an optical pulse 230 of a laserbeam is directed along a path 235 into the region 220 of the pulsesplitter 200 and at an incidence angle θ₀ relative to the interface 225.The interface 225, in this implementation, is a 50% reflectiveinterfacial coating. The optical pulse 230 is divided by the interface225 into two optical pulses that are directed along paths 240, 245, eachof these two optical pulses is divided again at the interface 225 afterreflecting off of either the top or bottom surface 205, 210. Fouroptical sub-pulses 250, 252, 254, 256 exit the pulse splitter alongpaths 260, 265, with a pair of pulses emerging from just beyond each ofthe two reflecting surfaces 205, 210, where the sub-pulses within eachpath have an intra-pair pulse spacing Δt of:

$\;{{{\Delta\; t} = {\frac{2}{c}\sqrt{\left( {n_{1}^{2} - n_{2}^{2}} \right)\left( {d_{1}^{2} - d_{2}^{2}} \right)}}},}$where c is the speed of light. Moreover, sub-pulses emerging along thesame path (either 260 or 265) are spatially overlapped if the incidenceangle θ₀ satisfies:

${{\cos\;\theta_{0}} = {\frac{d_{1}}{n_{1}}\sqrt{\frac{n_{1}^{2} - \overset{15}{n_{2}^{2}}}{d_{1}^{2} - d_{2}^{2}}}}},$a condition that can be met even with loose tolerances on d₁, d₂ if thepulse splitter 200 is mounted on a rotational stage.

In another implementation shown in FIG. 2B, a second optical pulse 270is directed along a path 275 into the region 215 of the pulse splitter200 near the reflecting surface 205. In this way, four more sub-pulses280, 282, 284, 286 can be created, where the sub-pulses 280, 282, 284,286 exit along the paths 260, 265 as shown.

Referring to FIGS. 3A and 3B, a pulse splitter 300 is designed based onthe principles discussed above with respect to FIGS. 2A and 2B. In thedesign shown in FIGS. 3A and 3B, the values of the refractive indices n₁and n₂ and the values of the thicknesses d₁ and d₂ for regions 315, 320,respectively, have been selected to produce output pulses having anintra-pair pulse spacing Δt of 37 ps.

The pulse splitter 300 is mounted in an optical holder 390 that isattached to a base 392 that is mounted on a motion (for example, arotational) stage 394. The holder 390, the base 392, and the rotationalstage 394 can be made of any suitable rigid material such as, forexample, aluminum or stainless steel or a non-conductive material. Themotion stage 394 can enable rotational movement of the base 392, theholder 390, and the pulse splitter 300 mounted to the holder 390 aboutan axis 396, and the motion stage 394 can also enable translationalmovement of the base 392, the holder 390, and the pulse splitter 300 inone or more directions.

The optical holder 390 includes input ports 317, 322 that are coupled tothe regions 315, 320, respectively, to permit the passage of the pulsesalong the respective paths 275, 235 through the holder 390 and into thepulse splitter 300. The optical holder 390 also includes output ports319, 324 that are coupled to the regions 315, 320, respectively, topermit the passage of the pulses along the respective paths 260, 265 outof the pulse splitter 300 and through the holder 390.

The holder 390 is designed with an internal cavity that is sized toreceive the pulse splitter 300. In the design shown in FIGS. 3A and 3B,the region 315 is made of a substrate 355 having an index of refractionn₁ greater than 1 and the region 320 is a cavity 360 that is filled withair having an index of refraction n₂ equal to 1. Thus, for example, thesubstrate 355 is made of a fused silica substrate. In the implementationshown in FIGS. 3A and 3B, the reflecting surface 305 is formed on a topsurface of the fused silica substrate 355 as a coating 365 that is fullyreflective at the wavelength of the light from laser, and the reflectingsurface 310 is formed as a separate mirror 370 that is placed at thebottom of the cavity 360. The coating 365 can be applied in a centralregion of the top surface of the substrate 355 and the surfacesurrounding the coating 365 can be either coated with an anti-reflectingcoating or can be uncoated to allow the light to pass through the topsurface of the substrate 355 along paths 275 and 260. Moreover, thepartially reflective interface 325 is formed as a coating 375 on abottom surface of the fused silica substrate 355.

The cavity 360 is defined between the bottom surface of the substrate355 and an internal surface 380 of the holder 300 and the upper surfaceof the mirror 370. The substrate 355 can be separated from the internalsurface 380 with a spacer (not shown) that is placed between the bottomsurface of the substrate 355 and the internal surface 380 and the mirror370, and the spacer is placed so that it does not interfere with thepaths 260, 265, 275, 235. In some implementations, a biasing device suchas a spring can be placed below the mirror 370 to bias the mirror 370against the spacer, which pushes against the substrate 355, which pushesagainst the optical holder 390.

Referring also to FIGS. 4A and 4B, a pulse splitter 400 is designedbased on the principles discussed above with respect to FIGS. 2A and 2B.In the design shown in FIGS. 4A and 4B, the values of the refractiveindices n₁ and n₂ and the values of the thicknesses d₁ and d₂ forregions 415, 420, respectively, have been selected to produce outputpulses having an intra-pair pulse spacing Δt of 74 ps.

As with the pulse splitter 300, the pulse splitter 400 is also mountedin an optical holder 490 that is attached to a base 492 that is mountedon a motion (for example, a rotational) stage 494. The motion stage 494enables rotational movement of the pulse splitter 400 about an axis 496,and the motion stage 494 can also enable translational movement of thepulse splitter 400 in one or more directions.

The optical holder 490 includes input ports 417, 422 that are coupled tothe regions 415, 420, respectively, to permit the passage of the pulsesalong the respective paths 275, 235 through the holder 490 and into thepulse splitter 400. The optical holder 490 also includes output ports419, 424 that are coupled to the regions 415, 420, respectively, topermit the passage of the pulses along the respective paths 260, 265 outof the pulse splitter 400 and through the holder 490.

The holder 490 has an internal cavity that is sized to receive the pulsesplitter 400. The region 415 is made of a substrate 455 having an indexof refraction n₁ greater than 1 and the region 420 is a cavity 460 thatis filled with air having an index of refraction n₂ equal to 1. Thus,for example, the substrate 455 is made of a fused silica substrate. Inthe implementation shown in FIGS. 4A and 4B, the reflecting surface 405is formed on a top surface of the fused silica substrate 455 as acoating 465 that is fully reflective at the wavelength of the light fromlaser and the reflecting surface 410 is formed as a separate mirror 470that is placed at the bottom of the cavity 460. The coating 465 can beapplied in a central region of the top surface of the substrate 455 andthe surface surrounding the coating 465 can be either coated with ananti-reflecting coating or can be uncoated to allow the light to passthrough the top surface of the substrate 455 along paths 275 and 260.Moreover, the partially reflective interface 425 is formed as a coating475 on a bottom surface of the fused silica substrate 455.

The cavity 460 is defined between the bottom surface of the substrate455 and an internal surface 480 of the holder 400. The substrate 455 canbe separated from the internal surface 480 with a spacer (not shown)that is placed between the bottom surface of the substrate 455 and theinternal surface 480 and the spacer is placed so that it does notinterfere with the paths 260, 265, 275, 235. In some implementations, abiasing device such as a spring can be placed below the mirror 470 tobias the mirror 470 against the spacer, which pushes against thesubstrate 455, which pushes against the optical holder 490.

Referring to FIG. 5A, the pulse splitter 200 (or the pulse splitters 300or 400) can be used in an optical arrangement 500 that produces as anoutput eight pulses that travel along path 505 from one input pulse ofthe laser beam that travels along path 510. The arrangement 500 includesa beam splitter 515 that divides the input pulse into two pulses thattravel along paths 235, 275. The pulse along path 235 is delayedrelative to the pulse along path 275 using, for example, mirrors 530,and the two pulses are then directed to the pulse splitter 200. Thesub-pulses 250, 252, 280, 282 and 254, 256, 284, 286 (not labeled inFIG. 5A) exit, respectively, along the paths 260, 265, and the pulsesalong path 260 (for example) are directed through a half-wave plate 535,and the pulses directed along both paths 260, 265 are directed through apolarizing beam splitter 540 to recombine the pulses into the commonpath 505. With appropriate delays, none of the eight pulses overlap intime, yielding a single output beam along path 505 that has a pulsetrain 550 shown in FIG. 5B and has an eight times higher repetition ratethan the pulse directed along path 510. The inter-pulse delay time Δt(the spacing in time between each pulse of the pair) is controlled bythicknesses d₁ and d₂ and the refractive indices n₁ and n₂ within thepulse splitter 200. Moreover, the separation between one pair of pulsesand another pair of pulses is given by the lengths of the beam paths235, 260, and 265. The inter-pair delay time 580 is controlled by thefree-space propagation distance between the beam splitter 515 and thepolarizing beam splitter 540.

Referring to FIG. 6, a monolithic pulse splitter 600 is designed togenerate ten equal, approximately equal, or unequal power sub-pulses(having a pulse train 680) from a single pulse 630 input into the pulsesplitter 600 along path 635. The pulse splitter 600 includes a pair ofparallel reflecting surfaces 605, 610 that define an internal openingthat houses a first region 615 and a second region 620. As with thedesign shown in FIGS. 2A and 2B, the first region 615 has a firstrefractive index n₁ and a first thickness d₁ and the second region 620has a second refractive index n₂ that is different from the firstrefractive index n₁ and a second thickness d₂ that is different from thefirst thickness d₁.

The pulse splitter 600 includes a partially reflective interface 625that is divided into segments 626, 627, 628, 629, 631 of varyingreflectance. The reflectances of the segments 626, 627, 628, 629, 631are selected to ensure that the output pulses emitted from the pulsesplitter 600 are of the same power. Or, the reflectances of the segments626, 627, 628, 629, 631 are selected to generate output pulse trainswith specific patterns in the power output.

In the implementation of FIG. 6, five pulses are generated along eachoutput path 660 and 665. The reflectance of segment 626 is 50%, thereflectance of segment 627 is 100%, the reflectance of segment 628 is67%, the reflectance of segment 629 is 60%, and the reflectance ofsegment 631 is 50%. The pulse 630 is input through region 620 andimpinges upon segment 626 of the interface 625 at {circle around (1)},where the pulse 630 is split into two equal power sub-pulses, which arereflected off the surfaces 605, 610 and then directed to segment 627 ofthe interface 625 at {circle around (2)}, where the sub-pulses arecompletely reflected (because the segment 627 has a 100% reflectance).The sub-pulses reflected from the interface segment 627 are thenreflected off the surfaces 605, 610 and then directed to segment 628 ofthe interface 625 at {circle around (3)}, where the sub-pulses are splitwith 67% of the power being reflected and 33% being transmitted throughthe segment 628. These four sub-pulses are reflected off the surfaces605, 610 and then directed to segment 629 of the interface 625 at{circle around (4)}, where they are split with 60% of the power beingreflected at the segment 629 and 40% being transmitted through thesegment 629, thus resulting in eight sub-pulses. These eight sub-pulsesare directed to the surfaces 605, 610, where they are reflected anddirected toward the segment 631 of the interface 625 at {circle around(5)}.

The eight sub-pulses are split at segment 631 such that 50% of the poweris reflected and 50% of the power is transmitted, and because of thetime delay between the eight sub-pulses, some of the reflected andtransmitted pulses overlap in time and result in ten equal-power subpulses 680, five of which are output along path 660 and five of whichare output along path 665.

Other reflectances can be used as long as the output pulses that travelalong paths 660, 665 have approximately equal power, if the applicationdictates. The ten pulses generated along output paths 660, 665 (andshown in the pulse train 680) are then appropriately delayed andrecombined, for example, using the setup shown in FIG. 5A.

In FIG. 6, the reflectance at the interface 625 is varied at eachreflection point to produce output pulses having substantially equalpower to achieve a balance between the signal and the photodamage thatresults when using the pulse splitter 600 in 2PFM. The reflectance atthe interface 625 at each reflection point is varied so that the overallenergy of each temporally overlapped pulse is equivalent to that ofthose pulses that remain temporally distinct. In this case, fivereflections of one input pulse at the interface 625 lead to 10 outputequal-energy sub-pulses.

Thus, in general, a pulse splitter can be designed to generate 2N pulsesfrom each output port for a single input pulse, and such a pulsesplitter is configured to provide N bounces or passes through thepartially reflective interface. For N greater than two, the reflectanceat a particular pass through the partially reflective interface may bedifferent than the reflectances at other passes through the partiallyreflective interface to ensure that the powers of all output pulses arethe same, if so desired. Moreover, a pulse splitter can be designed toproduce 2^(N−1) pulses at each output port, such a pulse splitter has Nregions of 50% reflectance (to split the pulses), interspersed with2^(N−1)−N totally reflecting regions (to temporally separate otherwiseoverlapping pulses), as outlined in FIG. 7.

The term “splitting ratio” is used below to refer to the number ofoutput pulses N produced relative to the number of input pulses, whichcan be by convention one. Thus, the “splitting ratio” is also the numberof output pulses N for each single input pulse.

Referring to FIG. 7, a pulse splitter 700 is designed to produce 16equal or substantially-equal power sub-pulses from one pulse 730 inputinto the pulse splitter 700 along path 735. The pulse splitter 700includes a pair of parallel reflecting surfaces 705, 710 that define aninternal opening that houses a first region 715 and a second region 720.As with the design shown in FIGS. 2A and 2B, the first region 715 has afirst refractive index n₁ and a first thickness d₁, and the secondregion 720 has a second refractive index n₂ that is different from thefirst refractive index n₁ and a second thickness d₂ that is differentfrom the first thickness d₁.

The pulse splitter 700 includes a partially reflective interface 725that is divided into segments 726, 727, 728, 729, 731 of varyingreflectance. The reflectances of the segments 726, 727, 728, 729, 731are selected to ensure that the output pulses emitted from the pulsesplitter 700 are of the same or substantially the same power. In thiscase, the reflectance of each of the segments 726, 728, and 731 is 50%and the reflectance of each of the segments 727 and 729 is 100%. Thus,pulses that impinge upon segments 727 and 729 of the interface 725 aretotally reflected at the interface 725.

In one implementation in which pulse splitting ratios of 2^(N) suffice(to enable 2PFM), it is possible to create output sub-pulses of equalpower using N regions of 50% reflectance (such as segments 726, 728,731), which split the pulse, and 2^(N−1)−N totally reflecting regions(such as segments 727, 729), which separate sub-pulses that wouldotherwise be temporally overlapped. Using only two interfacial coatingsof differing reflectance considerably simplifies the fabrication of suchpulse splitters.

With any of the pulse splitter geometries shown above, the arrangementof input and output beam splitters shown in FIG. 5A can be used to forma single input, single output pulse splitter of splitting ratio 4N or2^(N−1).

The pulse splitters shown and described above rely on the differentialoptical path length to cause the sub-pulses to separate in time fromeach other. Another phenomenon that tends to work against this conceptis, group-delay dispersion (GDD) within the materials internal to thepulse splitter and group-delay dispersion can lead to excessivesub-pulse broadening. The differential optical path length is thetemporal pulse separation Δt between the sub-pulses within the pulsesplitter and it is arises because the sub-pulses travel along differentpath lengths in the upper and lower regions of the pulse splitter, dueto the different thicknesses d₁, d₂ and the different refractive indicesn₁, n₂ of the regions. The sub-pulses can therefore be separated usingthis phenomenon. The differential optical path length is given by theproduct of the actual path length L and the refractive index of themedium n. Moreover, to minimize or reduce group-delay dispersion whileincreasing the differential optical path length (and hence the number ofsub-pulses N and/or their separation Δt), low dispersion fused silica(n₁=1.453 at 800 nm) and air (n₂=1) can be chosen for the respectiveregions 215, 220 of the pulse splitters. Other low dispersion materialscan be used for region 215.

Separation of the sub-pulses is important when using the pulse splitterfor MPFM or 2PFM, in which the temporal separation between sub-pulsesshould be greater than the time it takes for the molecule of thebiological sample to relax from a higher vibrational state to its lowestvibrational state of its excited state. In particular, a molecule in theground state absorbs the two photons from a first sub-pulse to reach thehigher vibrational state. The probability that a given molecule willsimultaneously absorb two photons is a function of both the spatial andtemporal overlap between the incident photons within the sub-pulses. Thenext sub-pulse should arrive after the molecule has relaxed to itslowest vibrational state of its excited state, and the next sub-pulsemay or may not arrive after the molecule has relaxed back down to theground state. Thus, the temporal separation between the two sub-pulsesshould be large enough to enable the molecule to relax to a lowestvibrational state of the excited state.

Nevertheless, group-delay dispersion sets a practical limit on both Nand Δt. For example, a pulse of 140 fs initial width (which is typicalof commercial Ti:Sapphire lasers) broadens to about 240 fs if thegroup-delay dispersion of the pulse splitter is 10⁴ fs², and suchgroup-delay dispersion corresponds to travel through a length L of about50 cm of low dispersion fused silica. However, for the pulse splitters200 and 600 of FIGS. 2A and 6, for example, that include input andoutput beam splitters 515, 540 as in FIG. 5A permitting both input portsof the pulse splitters to be used, the length L is related to N and Δtby:

${{N \cdot \Delta}\; t} = {\frac{4L}{c}{\left( \frac{n_{1}^{2} - n_{0}^{2}}{n_{1}} \right).}}$

Thus, a useful figure-of-merit is that N·Δt is approximately 5.1 ns fora fused silica/air pulse splitter exhibiting 10⁴ fs² of group-delaydispersion. In other words, there is a trade-off between the number ofsub-pulses output from the pulse splitter and their separation for agiven degree of dispersion. In the case of an input pulse having a 140fs initial width, the input pulse can be divided into 128 sub-pulses ofless than or equal to 240 fs width separated by 40 ps each, or into 512sub-pulses separated by 10 ps each. Furthermore, pulse broadening causedby group-delay dispersion decreases with increasing initial pulse width.Thus, in principle, pulse splitters can be designed for picosecond orlonger pulsed sources with N·Δt being approximately 500 ns or more. Inpractice, however, N is limited by the tight tolerances required on theparallelism of the various reflective surfaces (for example, betweenreflecting surfaces 205, 210, and 225) within the pulse splitter (forexample, the angle between two reflecting surfaces θ_(∥) should be lessthan or equal to about 50/N arcsec)—a constraint necessary for allsub-pulses to reach the same focal point within the sample to bemeasured.

Thus, although a single monolithic pulse splitter having about 50 outputpulses is within the capabilities of modern fabrication methods, a moremodular approach is shown in FIG. 8, which shows a system 800 thatrelies on a serial arrangement of delay lines and pulse splitters 805,810 each having smaller numbers N of output pulses and each having adifferent pulse spacing Δt. By adding or subtracting elements from thesystem 800, a different splitting ratio N may be obtained and therelationship between the signal and photobleaching could be betterstudied as a function of both the pulse repetition rate and the overallsplitting ratio.

The system 800 in FIG. 8 includes the two splitters 805, 810 of pulsespacing Δt=74 ps and Δt=37 ps, respectively, that each produce foursub-pulses for every input pulse. Thus, for example, the pulse splitter805 can be designed like the pulse splitter 400 of FIGS. 4A and 4B andthe pulse splitter 810 can be designed like the pulse splitter 300 ofFIGS. 3A and 3B.

The system 800 includes three 50%-50% beam splitters 860, 865, and 830and three 2.5 ns delay lines 867, 835, and 840. The delay lines 867,835, and 840 can be made, for example, by two reflecting surfacespositioned at opposite ends of a space so that a pulse that enters thedelay line is reflected a plurality of times from the reflectingsurfaces before exiting the delay line. The input pulse directed alongpath 825 is split by the beam splitter 860 into two beams, which areinjected into both input ports of the pulse splitter 805, and the beamsplitter 865 splits the beams emitted from the pulse splitter 805 intotwo beams, one of which is directed through the pulse delay 867, andthen both beams are injected into both input ports of the pulse splitter810. Thus, the second pulse splitter 810 receives the output pulses ofthe first pulse splitter 805, and 64 sub-pulses are produced along paths815, 820 from the original input pulse directed along path 825. Theoutput pulses along paths 815, 820 are directed through the beamsplitter 830, the pulse delays 835, 840, and then recombined using ahalf wave plate 845 and a polarizing beam splitter 850 to produce 128output sub-pulses directed along path 855, where the output sub-pulsesare arranged in thirty-two groups of four pulses, with an intra-groupspacing Δt=37 ps, distributed throughout the 12.5 ns interval betweenlaser input pulses (for a laser with a 80 MHz repetition rate). Byutilizing both input ports of each pulse splitter 805, 810, feeding 50%of each output from the pulse splitter 805 into each input of the pulsesplitter 810, and by adding additional delay lines 867, 835, 840, 128output sub-pulses can be produced covering several different timescales. The output pulse time sequence for the system 800 is shown belowthe diagram. Each vertical line in the sequence represents four pulseshaving an inter-pulse delay time of 37 ps.

The region 215 of the pulse splitter 805 can be a two-inch diameterfused silica plane parallel window having a thickness of about 0.5inches that is sold by CVI Laser (part number PW1-2050-UV) and theregion 115 of the pulse splitter 810 can be a two-inch diameter fusedsilica plane parallel window having a thickness of about 0.25 inchesthat is sold by CVI Laser (part number PW1-2025-UV). Each of thesewindows is custom-coated (by Reynard Corp.) with a 50% reflectivedielectric coating over the entirety of one face that acts as theinterface 225, and a greater than 98% reflective coating over a centralcircular region of the other face to form the reflecting surface 205,where the central circular region of the window used in the pulsesplitter 805 has a 0.6 inch diameter and the central circular region ofthe window used in the pulse splitter 810 has a 0.3 inch diameter. Bothcoatings exhibited constant reflectivity over the 780-910 nm wavelengthrange typical of 2PFM. However, the coatings can be fabricated to workover a much broader range covering, for example, an entire Ti-sapphirespectrum. Protected silver mirrors of diameter 0.5 inch for pulsesplitter 805 (purchased from Thorlabs, part number PF05-03-P01) and ofdiameter 0.28 inch for pulse splitter 810 (purchased from Thorlabs, partnumber PF03-03-P01) provided the bottom reflecting surfaces 210 of thepulse splitters 805, 810, respectively.

Precision ground spacers of thickness 0.28 inch (for the pulse splitter805) and 0.14 inch (for the pulse splitter 810) were used to positioneach mirror that provides the bottom reflecting surfaces 210 (such as,for example, mirrors 470 and 370) with respect its corresponding fusedsilica plane parallel window that provides the top reflecting surfaces205, thereby creating the air gap that served as the region 220 withineach of the pulse splitters 805, 810. With these gap dimensions,sub-pulses emerging from both output ports of each pulse splitter 805,810 were nominally spatially concentric at an input beam incident angleof 45°. In practice, each pulse splitter is mounted on a rotationalstage (that can be purchased from Thorlabs, part number PR01) tooptimize the overlap of all output beams such as shown in FIGS. 3A, 3B,4A, and 4B.

The non-polarizing beam splitter 860 can be purchased from Thorlabs(part number BS011), and the non-polarizing beam splitters 865 and 830can be purchased from Thorlabs (part numbers BS011, BS017,respectively). The half-wave plate 845 can be purchased from CASIX (partnumber WPA1312-λ/2-700 nm-1000 nm) or from Thorlabs, and the polarizingbeam splitter 850 can be purchased from Newport (part number10FC16PB.5). The three 2.5 ns delay lines 867, 835, 840 each consistedof opposed high-reflectivity (great than about 99.8%) low-dispersionmirrors (Precision Photonics Corp, MI1000-TiD) separated by a 4.0 inchspacer. Pulses traveled three round trips between these mirrors beforeemerging. Moreover, the greatest pulse separation occurred along thepath from the beam splitter 830 through the delay lines 835 and 840, andto the beam splitter 850.

The pulse splitter system 800 and design is structurally stable; afteran initial alignment, no re-alignment was needed over weeks ofoperation.

Removal of various elements from the system 800 shown in FIG. 8 leads tofive different test configurations, which are shown in FIGS. 9A-9E. InFIG. 9A, the system uses the pulse splitter 810 and has eight outputpulses (N=8) and a minimum pulse spacing of 37 ps (Δt_(min)=37 ps). InFIG. 9B, the system uses the pulse splitter 805 and has eight outputpulses (N=8) and a minimum pulse spacing of 74 ps (Δt_(min)=74 ps). InFIG. 9C, the system has eight output pulses (N=8) and a minimum pulsespacing of 1 ns (Δt_(min)>1 ns) but the system lacks any pulse splittersand uses only beam splitters 860, 865, and 830. In FIG. 9D, the systemhas 64 output pulses (N=64) and a minimum pulse spacing of 37 ps(Δt_(min)=37 ps). And, in FIG. 9E (as in FIG. 8), the system has 128output pulses (N=128) and a minimum pulse spacing of 37 ps (Δt_(min)=37ps). The power throughputs can be greater than about 90%, 50%, and 35%for 8×, 64×, and 128× pulse splitters, respectively.

Referring to FIG. 10, temporal sequences of pulses resulting from eachof the configurations shown in FIGS. 9A-9E, respectively, are shown.Thus, temporal sequence A corresponds to FIG. 9A, temporal sequence Bcorresponds to FIG. 9B, temporal sequence C corresponds to FIG. 9C,temporal sequence D corresponds to FIG. 9D, and temporal sequence Ecorresponds to FIG. 9E.

To explore the benefits of pulse splitting, the system of FIG. 8 wasused in conjunction with an 80 MHz Ti:Sapphire laser (Coherent Inc.,Chameleon) tuned to 910 nm and a commercial 2PFM (Prairie TechnologiesInc., Ultima) equipped with a 60×, 0.9NA water immersion objective(Olympus, LUMPlanFI/IR) to image fixed mouse cortical brain slicesexpressing cytosolic GFP in a subset of neurons.

Referring to FIGS. 11A-11I, in the first experiment, images were takenwith the pulse splitter system 800 (images in FIGS. 11A and 11D) andwithout the pulse splitter system 800 (images in FIGS. 11B and 11E) inits 64 pulse configuration (FIG. 9D). Identical energies of about 0.75pJ/pulse in the two cases were insured by delivering 64× more averagepower to the sample when the pulse splitter system 800 of FIG. 9D wasused. Corresponding comparative line profiles (graphs shown in FIGS.11C, 11F, and 11I) along the arrows shown at left images (in FIGS. 11A,11D, and 11G) and center images (FIGS. 11B, 11E, and 11H) (15 pixelaverages; orange or bottom in graph is with the splitter system, andgray or top in graph is without splitter system). Green arrows which arefound in the left images of FIGS. 11A, 11D, and 11G indicate smallneuronal processes that were more readily detected with pulse splitting.Measurement conditions of pulse splitting ratio (N), pixel dwell time,and average power at the sample are: for images in FIGS. 11A and 11D:64×, 0.4 ms, 3.9 mW; for images in FIGS. 11B and 11E: none, 25.6 ms,0.06 mW; for the image in FIG. 11G: 128×, 0.4 ms, 12.3 mW; and for theimage in FIG. 11H: none, 40.4 ms, 0.11 mW. The scale bars shown in thelower left portions of the images are: 20 mm in images of FIGS. 11A,11B, 11G, and 11H; and 2 mm in images of FIGS. 11D and 11E.

In the limit where the pulse energy delivered per pixel is sufficientlylow and the concentration of green fluorescent protein (GFP) issufficiently high for neither saturation to occur nor bleaching to besignificant, a 64× increase in signal rate should be observed with thesplitter system 800 of FIG. 9D provided that the output energy of eachsub-pulse with the splitter system 800 is the same as the energy of eachpulse without the splitter system 800. Comparable signals were obtainedin images of FIGS. 11A and 11D and in images of FIGS. 11B and 11E, eventhough the imaging speed of 0.4 μs/pixel with the splitter system 800(in images of FIGS. 11A and 11D) was 64 times faster than that withoutthe splitter system 800 (in images of FIGS. 11B and 11E). This alsoprovides indirect confirmation that the pulse splitter (and the system800) produced pulses of approximately equal intensity.

Tests with a 128× splitter system (as in FIGS. 8 and 9E) yieldedproportionally larger gains. For example, compare the image shown inFIG. 11G, which is an image taken with the splitter system of FIGS. 8and 9E, to the image shown in FIG. 11H, which is an image taken withoutthe splitter system of FIGS. 8 and 9E.

Another benefit of such fast imaging speeds is that less time exists forthe integration of background arising from ambient light, so that smallfeatures yielding weak intrinsic signals such as dendrites and dendriticspines (as shown in images of FIGS. 11D and 11E) are easier to identify(see, for example, the green arrows shown in graphs of FIGS. 11C and11I). Note the similarity in the resolution with and without the pulsesplitter (images of FIGS. 11D and 11E and the graph shown in FIG. 11F),which indicates that the pulse splitter system was effective inproducing spatially overlapped, co-propagating sub-pulses.

Pulse splitting reduces 2PFM photobleaching in fixed brain slices. Formany nonlinear optical processes, including 2PFM, photoinduced bleachingand damage increases even more rapidly with increasing excitationintensity I than does the signal generated. A simple model suggeststhat, in such cases, pulse splitting can be used to reduce the rate ofphotobleaching and photodamage at a given signal level. Thus, consider anonlinear process of signal S∝I^(α) and damage D∝I^(β), where β>α>1. Thesignal S₁ generated by one pulse equals the signal S_(N) generated by anN-pulse splitter when I₁ ^(α)=N(I_(N))^(α), or equivalently,I_(N)=N^(−1/α)I₁, where I₁ and I_(N) are the respective pulseintensities. In this limit, the photodamage D∝N(I_(N))^(β) with N pulsesis reduced to N^(1−β/α) times the damage D₁ with a single pulse. Toobtain the same signal rates in the two cases, the N pulses aredelivered at N times the original repetition rate, requiring an averagepower at the sample greater by a factor of N^(1−1/α).

Referring to FIGS. 12A-12C, to test this model, 2PFM line scans wereperformed with and without the pulse splitter (for example, the pulsesplitter system 800) in neighboring regions of the same GFP-labeled somawithin a fixed brain slice, and the rate of decay of the fluorescencesignal was recorded. A fixed brain slice was used for these initialmeasurements to avoid complications in the quantitative analysis ofphotobleaching caused by GFP diffusion. All five splitter configurationsin FIGS. 9A-9E were tested, covering output pulse numbers (or splittingratios) of N=8 (FIGS. 9A-9C), N=64 (FIG. 9D), and N=128 (FIG. 9E). Theaverage power PN required to achieve a constant signal rate scaled asthe square root of N (FIG. 12A), as expected for two-photon MPFM (thatis, N^(1−1/α)=√{square root over (N)} for α=2). This provided furtherindirect evidence of the uniformity of the pulse intensities provided bythe pulse splitter system 800. These results conformed the √{square rootover (N)} dependence expected of 2PFM (dashed line) in FIG. 12A.

In FIG. 12B, the graph shows typical data of the photobleaching signalversus time (the fluorescence over time relative to the fluorescence attime t=0), with 128× splitting (shown in the green or top set of data)and without 128× splitting (shown in the gray or bottom set of data). Tomeasure the photobleaching rates accurately, a single exponential decaymodel was insufficient, and thus a double-exponential decay formula,S=S₀+S₁e^(−t/T) ¹ +S₂e^(−t/T) ^(is 2) , was used to fit the data and isshown as the black line through the data of FIG. 12B. The shorter decaytime T₁ describes the precipitous drop of fluorescence intensityobserved at the beginning of the excitation, and T₂ depicts thebleaching on a longer time scale. For the green or top curve, theshorter decay time T₁=45 s and the longer decay time T₂=109 s. For thegray curve or bottom curve, T₁=11 s and T₂=53 s.

The ratio of the decay time with the splitter system 800 to that withoutwas measured for both T₁ and T₂ in all five splitter systems shown inFIGS. 9A-9E, and the results are summarized in FIG. 12C. Average powerwas about 1 mW at the sample without the splitter system 800, and wasabout √{square root over (N)} mW using the splitter system. Error barsrepresent standard deviations inferred from multiple measurements (N>5).The values of β are derived from these ratios and reflect theexponential dependence of the bleaching of fixed GFP on intensity (thatis, the photodamage D∝I^(β)).

In every configuration and on both time scales (T₁ and T₂), the rate ofphotobleaching at constant signal was reduced by inclusion of thesplitter system. The largest gains occurred for T₁ (for example, greaterthan 4× with the 128× splitter), a fortuitous result considering thatthe fast photobleaching component dominates most 2PFM imagingapplications at typical pixel dwell times. Furthermore, both T₁ and T₂continued to improve with increasing N up to the largest value tested(N=128) suggesting that additional gains can be obtained with evenlarger splitting numbers (where the number of output pulses N for everyinput pulse increases to above 128).

A concern during the design of the testing experiment was that pulsesplitters having sub-pulse separations (Δt) significantly shorter thanthe about the 3 ns fluorescence lifetime of GFP might actually result inmuch faster photobleaching than the laser alone, due to the possibilitythat 2PFM bleaching is dominated by the absorption of additional photonsfrom molecules already in the excited state. However, the relativeindependence of the bleaching ratio for both T₁ and T₂ when 8× splittersof Δt=37 ps, 74 ps, and greater than 1 ns were used suggests that thisconcern is not warranted for the power levels and GFP concentration usedin this study. Consequently, it should be feasible to develop pulsesplitters having significantly higher splitting ratios (that is, theratio of the number of output pulses, N, to the number of input pulseswhich is typically 1), since comparatively small (about 10-50 ps) pulseseparations Δt can be used, leading to large values of N beforeGDD-driven pulse broadening becomes significant.

Given the results in FIG. 12C, the relationship D_(N)/D₁=N^(1−β/α) fromabove, and the fact that α=2 for 2PFM, it is possible to estimate theexponent β that dictates the intensity dependence of the bleaching (thatis, D∝I^(β)) for both the fast and slow bleaching components and underall five splitter system scenarios shown in FIGS. 9A-9E. The ten values,shown in FIG. 12C, vary between 2.2 and 2.6. The fact that these datawere obtained from fixed GFP-labeled samples may explain why the valuesderived here differ from those reported elsewhere.

More examples of applications using the pulse splitters and systemsdescribed above are detailed below.

Pulse splitting reduces photobleaching in living C. Elegans larvae. Toassess if different photobleaching mechanisms predominate forfluorescence in living vs. fixed specimens, the effect of pulsesplitting on GFP photobleaching in muscle cells of C. Elegans strandPD4251 was also measured. C. Elegans larvae were paralyzed using 50 mM2,3-butanedione monoxime (BDM) and held stationary on 2% agar. Repeatedline scans were performed over individual muscle cells with and withouta 64× pulse splitter system (such as the system shown in FIG. 9D) untilthe fluorescence signal was largely depleted in each case. An averagepower 8× higher was used with the pulse splitter system to yield similarsignal rates in the two cases.

As shown in FIGS. 13A-D, images of each muscle cell were taken before(at t=0) and after bleaching (at t>0). The normalized photobleachingcurves measured from four cells under each experimental condition areshown in FIG. 13E. FIGS. 13A-13E show the effect of 64× pulse splittingon in vivo photobleaching within a GFP-labeled C. Elegans larva: withmuscle cells before (FIGS. 13A and 13C) and after (FIGS. 13B and 13D)GFP photobleaching. Cells subjected to bleaching both with (FIGS. 13Cand 13D) and without (FIGS. 13A and 13B) pulse splitting are marked withdashed white curves. FIG. 13E shows normalized photobleaching curves(the 2PFM fluorescence signal) obtained by repeated line scans oversingle muscle cells, fit with single exponential decay curves. Thecurves are “normalized” by defining the signal as one unit at the startof the first scan on a given cell. As the cell is line scanned moretimes, the fluorescence signal drops due to bleaching, as shown in thesecurves. However, the splitter signal drops less rapidly with repeatedscans, indicating less photobleaching with the pulse splitting. Thecurves obtained from the cells in FIGS. 13A and 13C are labeled withgray and green asterisks, respectively. The scale bars are 1 micron.

Because of the differences in cell size and GFP expression level, boththe initial 2PFM signal (that is, the signal at the initial time thelaser is directed to the sample) and photobleaching dynamics varied fromcell to cell.

The photobleaching curves in C. Elegans from FIG. 13E, except beforenormalization, are also shown with (FIG. 14A) and without (FIG. 14B) a64× pulse splitter. The initial 2PFM signal (at time 0) measured with(FIG. 14A) and without (FIG. 14B) pulse splitting was comparable andfell within a similar range.

The normalized photobleaching curves in FIG. 13E clearly demonstrate thebenefits of pulse splitting: a GFP-labeled cell was still visible after120 seconds with splitting (as shown in FIG. 13D), but anotherGFP-labeled cell was completely bleached after only 50 seconds withoutsplitting (as shown in FIG. 13B).

In contrast to the fixed slice measurements, each in vivo photobleachingcurve could be well-fit to a single exponential decay. The averagephotobleaching rate with 64× pulse splitting decreased by about 9-fold,from which a bleaching exponent β≈3 can be inferred, in agreement withprevious GFP photobleaching experiments in live cells. Thus, compared tothe fixed GFP case in FIGS. 12A-12C, the benefit of pulse splitting ismuch larger in living tissue, likely due to the higher concentration andmobility of oxygen in living specimens. These results also suggest thatthe effect of heating due to linear absorption on photobleaching in C.Elegans is minimal, since pulse splitting at an average power of 43 mWled to less bleaching than an average power of 5.5 mW without splitting.

Pulse splitting using the pulse splitters described above reducesphotodamage in functional Ca²⁺ imaging of hippocampal neurons in acutebrain slices. A particularly important application of 2PFM inneuroscience is imaging of neural activity using Ca²⁺ indicators. Inthis application, the Ca²⁺ indicator concentration is sufficiently highand its diffusion sufficiently fast that photodamage affects experimentsmore than photobleaching. During a typical experiment, the basalfluorescence level F increases with exposure time, and ΔF/F, therelative fluorescence intensity change upon neuronal activity, decreaseswith time. Prolonged exposure also often leads to severe morphologicalchanges, such that the viability of dendrites and spines undergoingmeasurement ultimately limits the quantity and quality of the collectedtraces. Here again photodamage is known to have a higher than secondorder dependence on excitation intensity, so pulse splitting shouldprove beneficial.

To test this, we performed Ca²⁺ imaging experiments on CA1 pyramidalneurons in 400 μm thick acute rat hippocampal slices at physiologicaltemperature. Neurons were filled with 200 μM Oregon Green BAPTA-1 usingwhole-cell patch-clamp pipettes. To ensure uniform filling andexcitation of proximal dendrites access resistance was kept minimal, aten to fifteen minute filling time was given before recording wasinitiated, all recordings were limited to dendritic regions within 150μm of the soma and separate dendrites within the same focal plain wereused for comparison. Two back-propagating action potentials were inducedby somatic current injection (700 pA, 10 ms) at 6 s intervals (0.17 Hz)and the Ca²⁺ transient in dendrites and spines was detected by rapidline scans (512 linescans at 333 Hz) using 830 nm excitation. We chosethe percentage fluorescence change ΔF/F as the metric of photodamage,and measured the dependence of ΔF/F on accumulated exposure time bothwith and without a 64× pulse splitter.

FIGS. 15A-15E show the effect of 64× pulse splitting on photodamageduring Ca2+ imaging of CA1 pyramidal neurons injected with Oregon GreenBAPTA-1. The scale bars are 1 μm in FIG. 15A and 5 μm in FIGS. 15D and15E.

FIG. 15A shows the temporal evolution of ΔF/F from two dendriticbranches of a single neuron in response to repeated action potentialinitiation, one measured with a 64× splitter system (the top circles)and one without (the bottom circles). Basal fluorescence images of thetwo branches taken at various times are also shown as insets in FIG. 15Awith the 64× splitter system images being shown above the graph and theimages without the splitter system being shown below the graph. Averagepowers of 60 mW and 5.7 mW post objective were used with and without the64× splitter system, respectively. The power used with the 64× pulsesplitter system was chosen intentionally high (for example, 60mW>√{square root over (64)}×5.7=45.6 mW) and yielded a higher basalfluorescence signal, as shown in FIG. 16A. Although this skews thephotodamage comparison in favor of the no splitter scenario, the pulsesplitter still drastically reduced photodamage.

Without pulse splitting (the gray and bottom circles shown in FIG. 15A),ΔF/F decreased rapidly after about 75 s exposure (50 traces), and after120 traces, the basal fluorescence increased about seven-fold (insetimages with gray borders below the graph of FIG. 15A), while ΔF/Fdropped to 10% of its original value. At this point, the experiment hadto be terminated to prevent killing the entire dendrite. In comparison,with the 64× pulse splitter system, the basal fluorescence increasedmuch more slowly (insets with green borders above the graph of FIG.15A): after over 700 s exposure (500 traces), ΔF/F dropped to only halfof its original value. Defining the Ca²⁺ imaging “lifetime” as thenumber of traces required for ΔF/F to decrease by one-half, we find thata 6× gain in lifetime is afforded by the use of a 64× splitter system(such as the splitter system shown in FIG. 9D), despite the higherinitial basal signal we used.

Referring to FIG. 15B, ΔF/F was measured by taking longer traces (6.28 straces every 17 s) and using higher average powers (66 mW with the 64×splitter system and 5.7 mW without the splitter system). Referring toFIG. 15C, ΔF/F was measured by using higher average powers of 100 mWwith the 64× splitter system and 12.6 mW without the splitter system.Both with and without pulse splitting, higher average power compared toFIG. 15A led to faster photodamage, as expected.

FIGS. 16A-16C show Ca²⁺ transient traces at the beginning of each seriesof traces used to measure photodamage during Ca²⁺ functional imaging(FIGS. 15A-15C). Average powers are: in FIG. 16A, 60 mW with 64×splitter, 5.7 mW without; in FIG. 16B, 66 mW with 64× splitter, 9 mWwithout; and in FIG. 16C, 100 mW with 64× splitter, 12.5 mW without.Considerably more signal was obtained in FIG. 16A with the splitter, dueto the use of more than 8× higher average power. Given similar basalfluorescence (FIGS. 16B and 16C), pulse splitting always improved theCa²⁺ imaging lifetime, with more dramatic gains occurring atincreasingly high power (greater than about 10× in FIG. 15B and greaterthan about 20× in FIG. 15C).

In FIGS. 15D and 15E, basal fluorescence images are shown, taken beforeand after Ca²⁺ imaging of the two sister branches used to obtain FIG.15B. After 10 traces without pulse splitting, the upper branch showedsignificant damage, as indicated by its much elevated basal fluorescence(orange arrows, in FIGS. 15D and 15E). After two more traces, a brightlyfluorescent vesicular structure appeared along the branch (inset, inFIG. 15E). In contrast, with pulse splitting, the elevation of basalfluorescence in the lower branch was only moderately higher even after80 traces, (green arrows, in FIGS. 15D and 15E) and ΔF/F was still abovehalf maximum. In line with the C. Elegans measurements (shown in FIGS.13A-13E), damage due to heating by linear absorption did not appear tobe significant up to 100 mW when the 64× pulse splitter was used.

The results above demonstrate the efficacy of pulse splitting forenhancing signal and reducing photobleaching and photodamage in 2PFM, asshown in the imaging apparatus of FIG. 1. The same principle that isshown in FIG. 1 can be applied to other multi-photon imaging methods,including those based on second harmonic generation (SHG), sum frequencygeneration, and coherent anti-Stokes Raman scattering (CARS). Themicroscope used in SHG and CARS is very similar to that shown in FIG. 1.For CARS, two input beams at different frequencies are used. Therefore,the apparatus 100 would include a plurality of pulsed laser sources 105.The pulse splitters 112 can be used simultaneously on a plurality ofpulsed laser beams 107 at different wavelengths. Moreover, the benefitsof enhancing signal and reducing photo damage can be found when thepulse splitters 112 are applied to linear optical phenomena such aslinear light scattering.

Another possible, advantage is that the pulse splitter spreads theexcitation energy across many time points within the original repetitionperiod of the laser, whereas pulse broadening methods still concentratethe energy of each original pulse in a single, albeit lengthened, pulse.Damage mechanisms based on ultrafast excited state kinetics maytherefore be eliminated or significantly reduced when using the splittersystem due to the fact that the time between sub-pulses is greater thanthe molecular excited state relaxation time.

To prepare the fixed brain slices used in the tests described herein,C57B16 mice were injected with AAV2/1-GFP virus at postnatal day 13 to15. Fourteen days later, animals were sacrificed and the brains wereextracted. Brains fixed overnight in 4% paraformaldehyde were washedthree times with phosphate buffer and then cut into 60 μm thick sectionsusing a vibratome (Leica, VT1000S). For long-term storage, such sectionswere mounted in Vectashield (Vevtor Laboratories).

To prepare the hippocampal brain slices used in the tests describedherein, transverse hippocampal slices (400 μm-thick) were prepared from8 to 12-week old Sprague Dawley rats as previously described. Rats weregiven a lethal dose of ketamine and xylazine, perfused through theascending aorta with an oxygenated solution just before death anddecapitated. Hippocampal CA1 pyramidal cells were visualized using anOlympus BX-61 microscope equipped with differential interferencecontrast optics under infrared illumination. Experiments were performedat physiological temperature (34-36° C.) in ACSF containing thefollowing (in mM): NaCl 125, KCl 3, NaHCO₃ 25, NaHPO₄ 1.25, CaCl₂ 1.3,MgCl₂ 1, and glucose 25; and was saturated with 95% O₂ and 5% CO₂.Current-clamp whole-cell recordings (somatic membrane voltage V_(m)=−65mV) from somata were performed using a Dagan BVC-700 amplifier in theactive bridge mode, filtered at 3 kHz and digitized at 50 kHz. Patchpipettes had a resistance of 2-4 MΩ when filled with a solutioncontaining (in mM): K-methylsulphate 120, KCl 20, HEPES 10, NaCl 4,MgATP 4, Tris 2, GTP 0.3, phosphocreatine 14 (pH=7.25). The seriesresistance was between 8-15 MΩ. Neurons were filled with 200 μM Oregongreen BAPTA-1 (OGB-1) (Molecular Probes, Eugene, Oreg., USA) and imagedusing a water immersion lens (60×, 0.9 NA, Olympus, Melville, N.Y.,USA). To allow for diffusional equilibration of the indicators, cellswere loaded with the dye for at least 10 min before the start ofexperiments. In all cells two action potentials were initiated by abrief current injection into the soma (700 pA for 20 ms).

The GFP expressing C. Elegans Larvae were prepared as follows. C.Elegans was grown on agar plates spread with E. Coli bacteria. Larvaewere transferred onto 2% agarose gel, paralyzed by 50 mM 2,3-butanedionemonoxime (BDM), and covered with a cover glass for imaging.

Experiments were performed on a commercial 2PFM microscope (PrairieTechnologies, Ultima) equipped with a 60×, 0.9NA water immersionobjective (Olympus, LUMPlanFI/IR). Laser power was measured after theobjective and controlled by a Pockels cell (Conoptics, Model 302).Photobleaching line scan measurements were carried out at 2 ms per 1.6mm line. The GFP imaging and bleaching measurements were performed at awavelength of 910 nm, whereas the calcium imaging experiments wereperformed at 830 nm.

All the above features can be combined with one another in any desiredmanner. Other aspects, advantages, and modifications are within thescope of the following claims.

In other implementations, the pulse splitter 200 discussed above isdesigned in a non-planar geometry, for example, an annular geometry inwhich the reflecting surfaces 205, 210 are rolled into each other toform annular first and second regions 215, 220. In otherimplementations, the reflecting surfaces 205, 210 can be multilayerdielectric films.

In other implementations, the pulse splitter 200 can be designed suchthat the reflecting surfaces 105, 110 are nonparallel with each other.In this way, the sub-pulses 250, 252 and 254, 256 would diverge fromeach other as they exit the pulse splitter 200, thus resulting in morethan one focus at the sample 115 to be imaged and creating a multi-focalimaging apparatus.

Referring to FIGS. 17 and 18, a pulse splitter 1700 or 1800 can bedesigned with more than one pair of reflecting surfaces. In thesedesigns, the pulse splitter 1700 or 1800 includes three pairs ofreflecting surfaces, with the distance between each pair being distinctfrom the distance between each of the other pairs in the pulse splitter.For example, the pulse splitter 1700 includes three pairs of reflectingsurfaces, with surfaces 1702, 1704 facing each other, surfaces 1706,1708 facing each other, and surfaces 1710, 1712 facing each other. Firstand second regions 1715, 1720 fill the space between the pairs ofreflecting surfaces and an interface 1725 is between the first andsecond regions 1715, 1720, as was discussed above. Similarly, the pulsesplitter 1800 includes three pairs of reflecting surfaces, with surfaces1802, 1804 facing each other, surfaces 1806, 1808 facing each other, andsurfaces 1810, 1812 facing each other. First and second regions 1815,1820 fill the space between the pairs of reflecting surfaces and aninterface 1825 is between the first and second regions 1815, 1820, aswas discussed above.

Referring also to FIG. 19, in another implementation of the pulsesplitter 112, a pulse splitter 1900 is designed with a plurality ofoptical fibers 1905 that can be of varying lengths, one or more fibersplitters 1910, and one or more fiber couplers 1915. Each fiber splitter1910 splits its input pulse (including the input pulse of the laser beam107) into sub-pulses. Intermediate fibers 1905 (those between the fibersplitters 1910 and the fiber couplers 1915) introduce different timedelays for each sub-pulse because they have differing lengths. The fibercouplers 1915 recombine the sub-pulses from the fibers 1905 into asingle output beam 120. In another implementation, optical waveguidescan be used instead of the fibers 1905 and the waveguides could becreated monolithically via microfabrication on a flat substrate,although dispersion may prove challenging, particularly at the pulsesplitting and re-combining sections. Dispersion in the pulse splitter1900 might be reduced by using hollow core photonic bandgap fibers asthe fibers 1905.

1. A pulse splitting apparatus comprising: a pulsed laser source thatproduces laser pulses at an input repetition rate and an input pulsepower; and a pulse splitter that receives the laser pulses from thepulsed laser source and outputs a plurality of sub-pulses for each laserpulse from the pulsed laser source, wherein the sub-pulses have arepetition rate that is greater than the input repetition rate and atleast two of the sub-pulses have power less than the input pulse power,wherein the pulse splitter comprises at least two different materials oneither side of a partially-reflective interface with each of thedifferent materials extending from the partially-reflective interface toan at least partly-reflecting surface and having different refractiveindexes, wherein the sub-pulses travel through the at least twodifferent materials such that each of the sub-pulses travel alongdifferent optical path lengths through the different materials to causea temporal separation between each of the sub-pulses, wherein theoptical path length depends on both the distance traveled through thematerial and the index of refraction of the material.
 2. The pulsesplitting apparatus of claim 1, wherein the pulsed laser source is aTi-Sapphire laser.
 3. The pulse splitting apparatus of claim 1, whereinthe pulsed laser source produces time-averaged output powers of severalWatts.
 4. The pulse splitting apparatus of claim 1, wherein the pulsedlaser source operates with an input repetition rate of about 70-80 MHz,a pulse width from about 10 fs to about 2 ps, and at a 650-1100 nmwavelength.
 5. A pulse splitting apparatus comprising: a pulsed lasersource that produces laser pulses at an input repetition rate and aninput pulse power; and a pulse splitter that receives the laser pulsesfrom the pulsed laser source and outputs a plurality of sub-pulses foreach laser pulse from the pulsed laser source, wherein the sub-pulseshave a repetition rate that is greater than the input repetition rateand at least two of the sub-pulses have power less than the input pulsepower, wherein the pulse splitter comprises at least two differentmaterials through which the sub-pulses travel such that each of thesub-pulses travel along different optical path lengths through thedifferent materials to cause a temporal separation between each of thesub-pulses, wherein the optical path length depends on both the distancetraveled through the material and the index of refraction of thematerial; one or more pairs of at least partly-reflecting surfaces thatface each other; a first region between the one or more surface pairsand having a first refractive index and a first thickness; a secondregion between the one or more surface pairs and adjacent the firstregion, and having a second refractive index that is different from thefirst refractive index and a second thickness that is different from thefirst thickness; and a partially reflective interface between the firstand second regions.
 6. The pulse splitting apparatus of claim 5, whereinthe pulse splitter outputs N sub-pulses for every one pulse input to thepulse splitter.
 7. The pulse splitting apparatus of claim 1, furthercomprising a recombiner that receives the plurality of sub-pulses fromthe pulse splitter and recombines the sub-pulses to form a sub-pulselight source that is directed to a sample.
 8. The pulse splittingapparatus of claim 1, further comprising a delay external to the pulsesplitter and through which at least some of the output of the pulsesplitter is passed.
 9. The pulse splitting apparatus of claim 1, furthercomprising a motion stage to which the pulse splitter is attached suchthat the pulse splitter can be moved relative to the path that the laserpulses travel along.
 10. The pulse splitting apparatus of claim 1,wherein the pulse splitter is a passive pulse splitter.
 11. The pulsesplitting apparatus of claim 1, wherein the plurality of sub-pulses areof equal or approximately equal intensity.
 12. The pulse splittingapparatus of claim 1, wherein the pulse splitter outputs the pluralityof sub-pulses using substantially all of the available power from thepulsed laser source.
 13. The pulse splitting apparatus of claim 1,further comprising: a sample placed in the path of a sample beam that isformed from an output of the pulse splitter; and a detector thatreceives a multi-photon fluorescence signal emitted from the sample. 14.The pulse splitting apparatus of claim 13, wherein the temporalseparation between sub-pulses should be large enough to enable amolecule of the sample to relax to a lowest vibrational state of itsexcited state that is reached after multiple photons have been absorbedby the molecule.
 15. The pulse splitting apparatus of claim 1, whereineach of the sub-pulses have a power that is less than the input pulsepower.
 16. The pulse splitting apparatus of claim 1, wherein therepetition rate of the sub-pulses varies.
 17. A method of pulsesplitting comprising: producing a pulsed laser beam having a repetitionrate and a pulse power; directing the pulsed laser beam into a pulsesplitter that creates a finite number of sub-pulses for each input pulseof the pulsed laser beam by directing sub-pulses through at least twodifferent materials on either side of a partially-reflective interfacewith each of the different materials extending from thepartially-reflective interface to an at least partly reflecting surfaceand having different refractive indices and along different optical pathlengths through the different materials to cause temporal separationbetween each of the sub-pulses, wherein the sub-pulses have a repetitionrate that is greater than the repetition rate of the pulsed laser beam,and at least two of the sub-pulses have power less than the pulse powerof the pulsed laser beam.
 18. The method of claim 17, wherein the pulsedlaser source is a Ti-Sapphire laser.
 19. The method of claim 17, whereinthe pulsed laser source produces time-averaged output powers of severalWatts.
 20. The method of claim 17, wherein the pulsed laser sourceoperates with an input repetition rate of about 70-80 MHz, a pulse widthfrom about 10 fs to about 2 ps, and at a 650-1100 nm wavelength.
 21. Themethod of claim 17, wherein directing the pulsed laser beam into thepulse splitter comprises directing the pulsed laser beam into a firstregion that is defined between first and second reflecting surfaces andhas a first refractive index and a first thickness such that the laserbeam travels through the first region and impinges upon a partiallyreflective interface between the first region and a second regionadjacent the first region, and is split at the partially reflectiveinterface, where the light that is reflected from the partiallyreflective interface strikes the first reflecting surface and any lightthat transmits through the partially reflective interface strikes thesecond reflecting surface.
 22. The method of claim 21, wherein the pulsesplitter outputs N sub-pulses for every one pulse input to the pulsesplitter and the partially reflective interface is configured to provideN passes or bounces of the light through the pulse splitter.
 23. Themethod of claim 17, further comprising recombining the equal intensitysub-pulses output from the pulse splitter to form a sub-pulse lightsource that is directed to a sample.
 24. The method of claim 17, furthercomprising directing at least some of the sub-pulses output from thepulse splitter through a delay external to the pulse splitter.
 25. Themethod of claim 17, further comprising adjusting a position of the pulsesplitter relative to the laser beam path.
 26. The method of claim 17,wherein the pulse splitter produces the output signal without usingexternal energy to act on the pulsed laser beam.
 27. A passive pulsesplitter comprising: one or more pairs of at least partly-reflectingsurfaces that face each other; a first region between the one or moresurface pairs, the first region having a first refractive index and afirst thickness; a second region between the one or more surface pairs,the second region being adjacent the first region, and having a secondrefractive index that is different from the first refractive index and asecond thickness that is different from the first thickness, or both asecond refractive index that is different from the first refractiveindex and a second thickness that is not different from the firstthickness; and a partially reflective interface between the first andsecond regions.
 28. The pulse splitter of claim 27, wherein the secondregion is defined as an open air-filled region between the pair of atleast partly-reflecting surfaces.
 29. The pulse splitter of claim 28,wherein the first region is a fused silica substrate having a firstsurface that includes a coating that forms one of the at leastpartly-reflecting surfaces and having a second opposite surface thatincludes a coating that forms the partially reflective interface. 30.The pulse splitter of claim 27, wherein the partially reflectiveinterface includes two or more segments of different reflectance. 31.The pulse splitter of claim 27, wherein the partially reflectiveinterface includes at least one segment that is 100% reflective.
 32. Thepulse splitting apparatus of claim 1, wherein the distance between oneof the partly-reflecting surfaces and the partially-reflective interfaceis different from the distance between the other of thepartly-reflecting surfaces and the partially-reflective interface suchthat the thicknesses of the different materials is different.